Salts of ethyl o-(cyclohexylamino-alkanoylamino)-benzoate

ABSTRACT

Novel anthranilic derivatives represented by the formula:   WHEREIN R, n, and X are as defined hereinafter, have been found to be especially effective anesthetic drugs for fish.

United States Patent Naito et al.

[ 1 July 4, 1972 [54] SALTS OF ETHYL O- (CYCLOHEXYLAMINO- ALKANOYLAMINO)-BENZOATE [72] Inventors: Taken Naito; Miyoshi Hirata; Sumiro 21 App]. No.: 12,424

[30] Foreign Application Priority Data Feb. 2i, 1969 Japan ..44/12593 [52] US. Cl. ..260/470, 260/471 R, 424/310 [51] Int. Cl ..C07c 143/52 [58] Field of Search ..260/470, 471, 471 R [56] References Cited UNITED STATES PATENTS 1,678,317 6/1928 Billeter et a]. ..260/470 1,690,705 11/1928 Seydel ..260/47l OTHER PUBLICATIONS Goldstein et al., Principles of Drug Action, Harper & Row I969), pgs. 206, 266. Epstein et al., J. Am. Chem. Soc. 79 5,814 (1957) Primary Examiner-Lorraine A. Weinberger Assistant Examiner-John F Terapane Attorney0bion, Fisher & Spivak [5 7] ABSTRACT Novel anthranilic derivatives represented by the fonnula:

wherein R, n, and X are as defined hereinafter, have been found to be especially effective anesthetic drugs for fish.

5 Claims, No Drawings BACKGROUND OF THE INVENTION mammals or warm-blooded animals cannot be similarly used for anesthetizing fish. This failure is due mainly to the fact that since fish belong to the familyof cold-blooded organisms, they must be anesthetized rapidly. The anesthetic must be soluble 1 pi f the [mention in fresh water or marine water, and it must be intense in its ac- This invention relates generally to novel anesthetic drugs yet m be harmless both fish f Moreover for fish and more particularly to novel anthranilic derivatives h f must be Cpable P weanng ofi completely represented by the following general formula: a relanvfily Short Penod of Wh1le certain aminobenzoic acid derivatives, such as t- (cyclohexyIamino-acetylamino)-benzoate which is the para -NH (CH2)..NH- H isomer of one of the species of the present invention, have been synthesized and their anesthetic effect on warm-blooded "COOK animals such as guinea pigs or mice have been reported by Elias Epstein (J.Am. Chem.Soc. 79 5,8l45,817), there has where R represents a lower alkyl group having one to three been no reported literature showing the anesthetic effect of carbon atoms, n is 1 or 2, and X represents a residual radical these materials on fish or cold-blooded animals. Moreover, of acids such as hydrochloric acid, nitric acid, sulfuric acid, the present invention is concerned with h or h ri phosphoric acid, acetic acid, succinic acid, or methanesuland it has been found that the para-derivative is not effective fonic acid. within the context of this invention.

2. Description Of Prior Aft l I SUMMARY OF THE INVENTIQN Trarispotiatlon of hve fish becoming everfmcrgasmg These and other objects have now herein been attained, and necesslty Wlth the deVekPmFm of fish i f It des lrab le an especially suitable anesthetic for fish provided, by the use that the fish be transported in as fresh condition as possible n of the novel amhranilic compound having the formula. order to satisfy the high degree of human taste. Moreover, in view of the now flourishing fish pet industry, it is desirable that the fish suffer as little damage as possible during the transpor- 110 O (CH )nNH H tation stage. -HX

In order to economically transport live fish or to readily --COOR treat various fish diseases, it would be desirable to anesthetize the fish so that they will require less ox en for survival. B this means, a greater number of fish cari be transported in Z wherem and have been dlscloeqabove' single container because of the decreased expenditure of ox- DETAILED DESCRIPTION OF PREFERRED ygen from the water. It was first thought that the fish could be EMBODIMENTS treated for this purpose by use of the same type of anesthetics T novel anthram11c esters of the present mvenuorf F known effective for mammals. For example, ethyl p- Prepared by {eactmg an Nhalogenoacylanmramhc 9 aminobenzoate (hydrochloride),ethyldiethylaminoethyl-plower alkyl ester more than 2 moles of cyclohexylamme aminobenzoate (hydrochloride) and the like were considered, a temperamrfi of from C to The P can f but these compounds were found to be inapplicable, since be converted into a Salt of y of the followmg aclfisi even their mineral acid salts are too sparingly soluble in water hydrochloric acid nitfic acid, Sulfuric acid; P P acid, to be dissolved at the various limits of concentration necessary acetic acid, succinic acid, of methanesulfonlc acidto provide the anesthetic effect. Furthermore, it is difficult to y 0'(cyclohexylaminoacetylamino)-benZate revive the fish using these types of compounds or their derivahydrochloride (n l, R C H X Cl) in (I). For example, tives. Urethane series compounds have also been used in the using yellowtail (Seriola Quinqueradata), the effectiveness of prior art for fish anesthesia, but they must be used at a fairly this compound was compared with ethyl 'rn-aminobenzoate high concentration to effect adequate anesthesia. The high (MS-222-Sandoz Co.,Ltd.), another commercially known fish concentrations necessary are an even more serious problem anesthetic,the results of this comparison is tabulated in Tables when it is considered that the urethanes are known to be car- I and II. In these Tables, the values X,n and R refer to the cinogenic. Accordingly, the use of anesthetics effective for structural formula (I).

TABLE I Anesthetic time in concentration of Fish tested (water temp.) Compound employed 10 ppm. 30 p.p.n1. 60 p.p.111. 100 p.p.111.

. Q MS-222 4nnn.,12sec l3n1in..5$ sec. Cumulus mums (25 "{C0mpd (l):11=l, l{=CgII5,X=CI hn1i|1.,37 see. 10 n1in., 3sec. 5111i11., 38sec.

Cwim -"-i o fg iT(i Tri i{ii iiinjii oijliiljljlijijjli1Iilifiiij iii'l'iiihlbi,ib'e'j tfiltti'aliii? I o C MS-222 16 min., 59 sec 2 min., 04 sec 0 min sec. mpho'lm (M {Compd. (I):11=1, R=C2H X=CL 6 min., 20 scc 2 min., 38 sec 1 min., 26 sec 0 111111., 49 sec.

TABLE II Anesthetic time in concentration of Fish tested (water temp.) Compound employed 5 ppmv 12.5 p.p.rn. 25 p.p.m. 50 p.p.m.

MS-222 8 min., 25 sec 5 min, 00 sec. Scriola macroccphalus (223 C.) Compd. (1): n=1, R=CZH5, X=Cl 1111111., 45 sec 1 nun, 20 sec 0 m1n., 40 secv Compd. (1): n=1, R=CZH5, X=CH3SO 2 mm., 15 sec 1 mm., 20 sec o i'zs 0 le nathus asciatus (22.8 Comp :n= 2 5 m 1n. sec m se p g f Compd. (1): n=1: R=CzH5I X=CH SO3 6 n11n.,'07 sec 2 m1n.: 42 sec BIS-222 8 min, 20 sec 3 n1in., 05 sec. Girclla punclata Compd. (I): 11 1, R=C2II5, X=Cl 1 min., sec 1 mm, 13 see 0 mm, 45 sec. Compd. (1): 11 1, R=C3l[5, X=CH S0 3111111.,15 sec 1 n1111., 48 sec 1111111., 01 sec.

This data was obtained by the following procedure: The fish were immersed in a solution containing various concentrations of each compound. The anesthetic time was represented by the required time for anesthesia after immersion. Anesthesexamples which are provided herein for purposes of illustration only and are not to be considered as being limiting in any manner.

EXAMPLE 1 (Synthesis) ia was considered that point at which the fish became 5 To 50 ml of benzene g of ethyl o-(B-chloropropionyhcompletely immobilized or remained relatively motionless. amino)-benzoate was dissolved and 12 g of cyclohexylamine Following anesthesia, the fish were transferred into water was added- The mixture was refluxed for 4 hours, cooled to without the drug and complete recovery was recognized. room temperature, and cyclohexylamine precipitate was fil- The various isomers (ortho-, meta-, and para-) of ethyl otered off. The filtrate was concentrated to a syrup in vacuo (cyclohcxylaminoacctylamino)-benzoate hydrochloride were 10 n h syr p w i olv in hlor form The chloroform also compared and the results tabulated in Table III. solution was ashed wi h water and dried with sodium s lfate.

TABLE III lAnesthetic time and other properties] Ortho-deriv. in above formula Metha'deriv. in above formula Para-deriv. in above formula Anesthetic time,

In concentration of 100 ppm.

12 min., 32 sec. 5 min., 00 sec 8 min,, 17 sec 12 min., 45 sec Not ones- 7 min., 08 see 3 min., 35 sec 19 min., 45 sec. 6 min., 500.. Net anesthe- 7 min., 45 sec. thetized.

7 min, 08 sec.

tized.

Solubility (g./ml.). Recovery time (minu Toxicity on mouse. (Llm: rug/kg.)

Fish tested: (iOid fish (Carassius auratus), temperature: iii-25 C.

As can be seen from Table iii, the ortho-derivatives of the series are shown to be the most effective from the point of view of rapid anesthesia within a short time period and from the point of view of rapid recovery of the fish to normal activity.

in general, it has been considered that the higher the anesthesia potency, the stronger is the toxicity. However, using the compounds of the present invention, judged by the brief recovery period shown in the experimental data for the ortho-derivatives, this generalized statement, which is so frequently applicable to warm-blooded animals, is not applicable to fish. The tables also show that the recovery time using the metaor para-derivatives of the same series is several times as long as that using the ortho-derivatives. It has also been observed that in the case of metaor para-derivatives, the fish become violently agitated at the recovery from the anesthesia. These experimental results lead us to the conclusion that only the ortho-derivative is suitable as an anesthetic drug for fish.

The suitable concentration of the present compound in its application varies depending upon the type of fish, the body weight of the fish, and the temperature of the water. Usually, concentrations of between 10 ppm and 100 ppm provide sufficient anesthesia, yet permits rapid recovery when the fish are removed from the drug. This low concentration is especially significant in view of the fact that MS-222 requires 50 ppm to 500 ppm and those of the urethane series requires 5,000 to 50,000 ppm.

in using the drug compounds of the present invention, any suitable method for application such as direct spraying, bathing or immersing and direct administration can be used depending upon the specific type of fish and the body weight.

Another advantage of the compounds of this invention is that their solubility is sufficiently high, both in fresh water and in marine water for wide practical application. The hydrochloric acid salt, as well as the other acid salts, are rapidly dissolved in both fresh water or marine water, and their solutions are harmless to both fish and man.

Synthesis of the present compounds can be easily accomplished by known processes. For example, ethyl 0- (B- cyclohexylamino)propionyl-amino)-benzoate (hydrochloride) can be prepared by reacting ethyl ortho-(flchloropropionyl)amino)-benzoate with cyclohexylamine in benzene, followed by treatment with an alcoholic hydrochloride.

Having generally described the invention, a further understanding can be obtained by reference to certain specific Methyl o-( N-cyclohexylaminoacetyl-amino )-benzoate hydrochloride M.P. l 74l 76 C (decomposed) Ethyl o-( N-cyclohexylaminoacetylamino)-benzoate hydrochloride M.P. 232-235 C Ethyl o-( N-cyclohexylaminoacetylarnino )-benzoate methanesulfonate M.P. l43145 C Ethyl o-(N-cyclohexylaminoacetylamino)-benzoate (Oily Product) EXAMPLE 2 To two liters of water treated with ion-exchange resin, 60 mg of ethyl o-(N-cyclohexylaminoacetylamino)-benzoate hydrochloride was added and 5 gold fishes (Carassius auratus) having 5 g of average body weight were added to the water. All the fish were anesthetized to lie still within 30 seconds to 2 minutes after immersing. When those anesthetized fishes were transferred into fresh water without the drug, the fish were revived within 2 minutes.

EXAMPLE 3 To two liters of water treated with an ion-exchange resin, 200 mg of ethyl o-(N-cyclohexylaminoacetylamino)-benz0ate hydrochloride was added and 5 common carps (Cyprinus carpio) having an average body weight of 7 g. were added to the water. All the fish were anesthetized 5 minutes after immersion. When transferred into fresh water without drug they were revived within 2 minutes.

EXAMPLE 4 Into 6 liters of artificial marine water was dissolved 500 mg of ethyl o-(N-cyclohexylaminoacetylamino)-benzoate ferred into fresh artificial marine water without the drug, they were revived within 5 minutea.

EXAMPLE 5 Into two liters of artificial marine water, 60 mg of ethyl o- (N-cyclohexylaminoacetylamino)-benzoate hydrochloride was dissolved and 5 pufi'ers (Fugu niphobles) weighing 2 to 3 g of body weight were added thereto. All the fish were anesthetized within about 2 minutes after immersion. When the anesthetized fishes were transferred into fresh artificial marine water, they were revived within 4 minutes.

Having fully disclosed the invention, it will be apparent to one of ordinary skill in the art that many changes and modifi cations can be made without departing from the spirit or scope thereof. Accordingly,

What is claimed and intended to be covered by letters patent is:

l. A compound represented by the following formula:

-- COOK 

2. The compound of claim 1 which is Ethyl o-(cyclohexylaminoacetyl-amino)-benzoate hydrochloride.
 3. The compound of claim 1 which is Ethyl o-(cyclohexylaminoacetyl-amino)-benzoate methane-sulfonate.
 4. The compound of claim 1 which is Ethyl o-( ( Beta -cyclohexylamino) propionylamino)-benzoate hydrochloride.
 5. The compound of claim 1 which is Ethyl o-(cyclohexylaminoacetyl-amino)-benzoate. 